Cervidae antlers exploited to manufacture prehistoric tools and hunting implements as a reliable source of ancient DNA

Antler is one of the primary animal raw materials exploited for technical purposes by the hunter-gatherer groups of the Eurasian Upper Palaeolithic (UP) all over the ecological range of deers, and beyond. It was exhaustively employed to produce one of the most critical tools for the survival of the UP societies: hunting weapons. However, antler implements can be made from diverse deer taxa, with different ecological requirements and ethological behaviours. Identifying the antler's origin at a taxonomic level is thus essential in improving our knowledge of humans' functional, practical and symbolic choices, as well as the human-animal interface during Prehistoric times. Nevertheless, palaeogenetics analyses have focused mainly on bone and teeth, with genetic studies of antler generally focused on modern deer conservation. Here we present the results of the first whole mitochondrial genome ancient DNA (aDNA) analysis by means of in-solution hybridisation capture of antlers from pre-Holocene archaeological contexts. We analysed a set of 50 Palaeolithic and Neolithic (c. 34-8ka) antler and osseous objects from South-Western Europe, Central Europe, South-Western Asia and the Caucasus. We successfully obtained aDNA, allowing us to identify the exploited taxa and demonstrate the archaeological relevance of those finds. Moreover, as most of the antlers were sampled using a minimally-invasive method, further analyses (morphometric, technical, genetic, radiometric and more) remain possible on these objects.

Antlers are an exoskeletal appendage characteristic of the Cervidae (deer) family with a yearly cycle of growth, fall and regrowth [38][39][40].Shape and size are highly variable between species; thus, their morpho-structural properties are very diverse [41][42][43].Such properties undoubtedly restrict their potential technical exploitation.Manufacturing a projectile point requires a fragment of antler that is both long and straight to provide the projectile with symmetry and enough thickness to ensure its solidity and right trajectory.Only developed antlers, specifically the beam parts, of adult individuals from some species can fulfill such requirements [6].This is likely the reason why some deer documented in several archaeological Pleistocene sites, like European fallow deer (Dama dama) and roe deer (Capreolus capreolus), never seem to be used for the production of hunting weapons or other tools.Roe deer antlers are generally unsuitable for technical exploitation due to their small size, and the low thickness of compact bone tissue [44].The European fallow deer, contrary to the Persian subspecies [28], has flat antlers with a less developed beam.Additionally, cervidae taxa occupied various habitats, from open landscapes to closed forests, swamps, and arctic tundra, and from mid to high latitudes, spanning the Eurasian mega continent during the late Pleistocene [45][46][47][48][49][50][51][52][53][54][55].Thus, both the morpho-structural properties and the ecological distribution of deer limit raw material availability in a given spatio-regional context.Nevertheless, practical constraints don't always explain the choices of Prehistoric societies.We have recent examples of cultural preferences inferred from the selection of one or several taxa [30,[35][36][37].Selecting certain species and anatomical parts has demonstrated both shared and divergent choices regarding the aesthetic-cum-symbolic set of personal ornaments and decorated bones from Western Europe and the Levant in the Early UP [35,36].
For the majority of antler objects, the designation of raw material is performed by macroscopic analysis [4].Categorizing osseous tissues' exact taxonomic origin is, however, generally only possible using biomolecular methods, albeit some attempts by X-ray micro-tomography have been made [56] to differentiate between red deer and reindeer antlers.A major difficulty lies in identifying the intensely transformed anatomical blank during the objects' production, involving the loss of many, if not all, specific diagnostic attributes.
Biomolecular techniques have therefore become invaluable tools to identify the species of raw materials used.Two methods can be employed for this purpose, namely palaeogenomics and palaeoproteomics (through Zooarchaeology by Mass Spectrometry method or "ZooMS").The latter method uses peptide mass-fingerprinting of collagen to identify the species of osseous fragments.It is widely used  in archaeology and palaeontology, with an expanding range of applications.ZooMS was first proposed by Buckley et al. [57] as a method for identifying the species of bone fragments where no morphological indicators are present.It was further developed [58,59] and recently applied for bone tools taxa identification following a non-destructive sampling technique [30].ZooMS is less invasive and cheaper than aDNA analyses.However, it only allows for discrimination at the family level, and therefore not always accurate enough to identify diverse deer taxa.Conversely, aDNA can provide more accurate data, potentially including the sex and the phylogeny of the exploited species even with little preserved aDNA, something impossible with ZooMS.Ancient DNA can therefore provide unique information about the makers/users of (pre)(historic) bone tools, and even potentially the prey hunted with a single antler projectile.Nevertheless, despite the importance of the diverse skeletal tissues for prehistoric past societies, palaeogenetics and palaeoproteomics analyses of osseous objects have mainly focused on bone [30,32,33,60] and tooth [34] artefacts.Genetic studies of antlers are mostly restricted to modern specimens in the context of deer conservation (e.g., Refs.[61][62][63][64][65]).Ancient DNA (aDNA) analyses, sometimes in combination with palaeoproteomics, of deer antlers have been restricted to palaeontological sites [65,66].Analyses of antlers from archaeological contexts have so far been limited to post-Pleistocene periods (Holocene context [67], pre-Viking contexts from Scotland and Scandinavia [68] and Middle Ages [69]. Here, we present the results of the ancient DNA (aDNA) analysis of a set of antler fragments and objects from Palaeolithic and Neolithic archaeological contexts (c.34-8ka.).These come from a range of sites in South-Western Europe (France, Spain), Central Europe (Austria, Czech Republic), South-Western Asia (Lebanon and Israel), and the Caucasus (Georgia) (Table 1).We obtained aDNA through a minimally invasive sampling method, allowing us to identify the exploited taxa, demonstrating that ancient antler objects can be a reliable long-term source of aDNA.In addition to the antler objects, the method was also applied to a series of bone tools enabling the comparison between antler and cortical bone.The method is combined with a custom-created set of capture baits for the mitochondrial DNA of 52 mammalian species (Supplementary Table 1), based on the most representative taxa of the Eurasian studied regions and the primary sources of human industry.The obtained mitochondrial data have been used to identify the exploited taxa and further explore five individuals' phylogenies.We quantitatively assess the invasiveness of our new method on the objects by studying their macro-morphology and structure to be able.Macroscopic and microscopic assessments, and as micro-CT scans confirmed that the macro and micro-morphology of objects remains broadly unchanged after sampling, allowing the carrying out of a range of further studies on the objects after sampling, including morphometric, technical, genetic, and radiometric analyses.

Results
We captured mitochondrial DNA from 50 bone and antler items.For 34 of those (72 %), we were not able to identify any nonhuman mammalian mitochondrial DNA.For seven of those samples (14 %), the species identified contradicted the preliminary visual analysis, which suggested that the items were made of antler, but the genetically identified species did not possess such exoskeletal appendages (Sus scrofa, Bos taurus, and Capra hircus).These results can be explained by the conservation of the items in the museum.These three species are consistent with those used to make animal-based glues, commonly used in museum conservation [70].Finally, for 17 of the items (34 %), it was possible to confidently identify the source species.While most of these yielded a very low mitochondrial coverage (<6x), 7 yielded more data (5 of which were made of antler), enabling further phylogenetic analyses.We checked the deamination values of the human DNA by selecting the human-aligned reads using samtools and checking the deamination values using mapdamage 2.2.1 [71].In any case, these values were greater than 0.01, suggesting the absence of substantial ancient human DNA in the samples.Out of the 28 antler items tested, 8 (29 %) gave enough results for a positive taxon identification.Bone samples were successful in 10 out of 20 (50 %).This confirms that antler is indeed a reliable source of aDNA, albeit not as efficient at preservation as bone (Fig. 2).
For the five samples for which extracts were obtained using both the traditional powdering method and the minimally-invasive method (Table 3), species identification was possible in two cases.Although the powdering method yielded a higher number of reads and a consequently higher coverage, the ability to identify species seems to be similar with both methods.It may therefore be recommended to use the powdering method in borderline samples, but the minimally invasive method seems to perform well enough when aDNA is fairly well preserved.

Discussion
Osseous tools are a fundamental proxy for understanding the subsistence and cultural networks of Palaeolithic peoples [31,72].The correct determination of the species origin is fundamental to gaining insights into the origin of the raw material employed, especially in areas where no or very few exploited animals are present [6,56].Recently, proteomics, especially ZooMS, has emerged as a reliable method to identify the taxon of animal-made artefacts with very little input material required [30].Despite huge recent improvements, ZooMS only enables the identification of taxa, without enabling further phylogenetic inference, and may lack resolution at the species level.Consequently, ancient DNA appears as a reliable tool to address questions regarding the exploitation of bone-tools in prehistory, when the key questions relate to species-origin and the possible existence of genetic similarities relating to trading networks.Previous work on personal ornaments has demonstrated that it is feasible to obtain the DNA of the wearers/makers of the ornaments [34].It is therefore also envisageable to recover the DNA and identify the preys hunted with osseous weapons, something which, to this day, remains subject to speculation.
It is well-accepted that ancient DNA preservation is related to climatic conditions, and that recovering DNA from warm and humid climates is particularly challenging [73].Here we observe that none of the four here-studied artefacts from the Middle East (namely those from Nahal Rahaf and Ksâr 'Akil) have yielded DNA-based taxonomic identification.In contrast, for 8 out of 15 from the Cantabrian region, this was possible.Therefore, it is clear that the success of the presented project is strongly determined by the environmental conditions determining the preservation of aDNA.In this study, however, we bring a new insight into sample efficiency related to the storing conditions and manipulation.Focusing on the temperate region samples, we observe that while 9 out of 31 pieces stored in collections yielded results.More importantly, the samples kept in museums for over 100 years and handled abundantly failed systematically (this includes all samples from the French collection).The items from Mladec were also stored and handled for a long time period prior to analyses.These seem to have been treated with animal-based glues at some point in their conservation history, as reflected in the taxa identified through the aDNA analysis.In stark contrast, the recently excavated samples from Tito Bustillo and Chufín (both caves in a temperate region) yielded excellent results, thereby confirming the suitability of pre-historic antler implements as a source of aDNA.
The unique status of Cervidae in Late Pleistocene hunter-gatherer societies is further reflected by the finding of many personal ornaments made from perforated red deer teeth [74,75], and through the frequent representations of red deer in Southeast European parietal and portable art [76][77][78], as well as that of other cervid species [79,80].Due to the diversity of the various cervid species' antlers' availability and technical constraints, there is high value in identifying the selected taxa in each region, site and layer, thereby allowing us to differentiate ecological and technological choices against the cultural selections of our Prehistoric ancestors.Nevertheless, we can only build an objective database solid enough on exploited deer taxa for technical purposes by applying aDNA analyses.Here we have successfully recovered phylogenetic information from 5 Iberian implements plausible with the use of local Cervus elaphus.Although biomolecular analyses are of great value, recent studies stress the significance of evaluating potential effects of various sampling methods on bone tools [32,81,82].Our study demonstrates that the minimally-invasive aDNA method implemented by Harney et al. [83] for human teeth (itself a modification of [84]) can be adapted and applied to bone and antler tools, when sampling by powdering is not possible.Another minimally-invasive method has recently been presented by Essel et al. [34].The team has successfully extracted aDNA of the raw material animal as well as that of the users/makers of the object, with no significant observable morphological modifications to the object itself.However, it must be noted that the time and equipment required to perform such extractions make it extremely challenging to perform in any environment outside the laboratory.In contrast, using our here-presented method enabled us to perform most of the extractions directly at the storage location of the item or even at the site itself, thereby not requiring the transportation or export of any items which can be necessary in some cases, especially when studying rare items that may not be possible to remove from collections.
The oldest antlers to yield aDNA so far came from Palaeontological contexts of around 12ka [65].Our study extends this range significantly, setting the stage to improve our knowledge of Upper Palaeolithic societies from the earlier H. sapiens groups permanently settling in Eurasia (C.45,000 years ago) to recent Prehistoric times.Our results demonstrate that pre-Holocene antler implements can be a source of aDNA.While bone and teeth have, thus far, been the primary tissues used to obtain aDNA, we hereby confirm worked antler as another potential source.Given the importance of antlers as a raw material for the hunter-gatherer groups at the end of the Pleistocene, but also for later societies up until the Middle Ages, it is critical to obtain from them as much data as possible by combining archaeological and biomolecular methods.

Material and methods
The analysed assemblage comprises 50 Upper Palaeolithic items encompassing hunting implements (projectile points and one harpoon), blanks, production wastes and domestic tools (awls) (Table 1).All items were studied with the full permission of the respective curators and collection caretakers.

Ancient DNA
DNA sampling was performed using two methods.Around ~50 mg of powder from the object's interior for some pieces were collected by drilling.The DNA was then extracted from powder following the protocol outlined by Ref. [85] with modifications described in Ref. [86], namely the replacement of the Qiagen Minelute column custom constructions for DNA purification with columns from the Roche High Pure Viral Nucleic Acid kit.Most items were sampled using the minimally-destructive extraction procedure presented here.It is based on the protocol described by Ref. [83] with several modifications detailed below.
The extractions were performed at the location of sample storage and inside the cave in the case of La Garma (Spain).The environment in which it was served was cleaned as thoroughly as possible: surfaces were wiped with a dilute (about 1.2 %) bleach solution and covered with a bleach-cleaned aluminium foil.We verified that no PCR was ever performed in the same space to avoid potential contamination.
The first step consisted of cleaning each object by wiping with a bleach solution (about 1.2 %) and then rinsing thoroughly with absolute ethanol.The pieces were then exposed to short-wave (254 nm) UV light for 10 min on each surface.
Unlike the procedure described in the Harney et al. protocol [83], the samples were not wrapped in Parafilm, but entirely submerged in extraction buffer.The exception was the samples stored at the Musée d'Archéologie Nationale (France), where the pieces were wholly wrapped in parafilm except for leaving a small window exposed (~2-4 cm 2 ).The smallest possible container was selected to fit the whole piece comfortably with as little spare space as possible.The possible containers were 5 ml, 15 ml and 50 ml Eppendorf DNA LoBind tubes and sterile plastic bags.
In some cases, the object was submerged for 20 min in extraction buffer, for a pre-digestion.The initial lysate was then discarded to remove the potential external DNA contamination.This was only performed for the later batch of samples containing the items from Chufín, Tito Bustillo-Área de Estancia, Nahal Rahaf 2 and Satsurblia (experimental items).The items were then re-submerged in an extraction buffer, the volume of which was adapted for each piece.The minimum amount that enabled the pieces to be fully submerged ranged between 1.0 and 15.0 ml.The extraction was performed in room-temperature to warm conditions at ~35 degrees C, with the liquid in the tubes moved around gently at regular 15-min intervals, while monitoring the effect of the buffer on the piece's surface condition.The duration of the extraction was adapted for each item.In all cases it was stopped at the latest as soon as any effect of digestion on the piece became visible or evidence of significant dissolution was detected through a marked change in the colour of the extraction buffer, as it is unfortunately not possible to objectively measure the lack of damage.Individual digestion times are given in Table 2, ranging from 0.5 to 2.5 h.The resulting lysate was then stored in a freezer.
These lysates were then brought to the ancient DNA laboratory at the University of Vienna, and further processed in a dedicated ancient DNA clean room.The lysate clean-up was performed following Dabney et al. (2013) [85] with the modifications described in Harney et al. (2021) [87].As most samples resulted in more than 1 ml of extraction buffer, a ratio of 13:1 was used to calculate the amount of binding buffer required for optimal binding of the DNA to the silica columns, and the entire mixture ran through the same column.
Subsequently, double-stranded libraries were built from 25.0 μl of extract, according to Meyer and Kircher [88].Qiagen MinElute PCR Purification kits were used for the intermediate clean-up steps.The libraries were double-indexed and amplified with the NebNext Q5U Master Mix DNA Polymerase (NEB) using a number of cycles calculated employing the qPCR analysis of 1 μl of the library.Indexed libraries were captured using a custom built capture kit for the mitochondrial DNA of 52 mammalian species (Supplementary Table 1).This capture kit has been designed by the team in Vienna and produced by myBaits (Arbor Biosciences) (table SI1).This capture kit allows screening for an extended list of species simultaneously, extending the possibilities to recover aDNA and improving the discrimination capabilities, allowing species-specific hits and better discriminating between species from the same family.This was then shallow-sequenced as part of a larger pool of samples on a single lane of a NovaSeq SP system.

Bioinformatics
Sequenced reads were processed after demultiplexing.Sequenced adapters and short reads below 30 were discarded using Cutadapt 4.2 [89].The remaining reads were aligned against 40 representative mammalian species in a competitive mapping (list) with bwa aln 0.7.17 [90], disabling seeding and with a gap penalty open of 2. The aligned reads were filtered by quality with samtools 1.16.1 [91], setting minimum mapping quality of 30 and removing duplicates with Picard-tools 2.27.5 [92].The remaining reads were inspected with mapdamage 2-2.2.1 [71] to determine the deamination patterns and with qualimap 2.2.1 [93] to inspect the results of the competitive mapping.Non-human species were considered positively identified when more than 50 reads could be mapped to the genome of a particular species.When more than one hit was present per sample, we focused on the dominant taxon (the one with the most mapped reads).We therefore considered this as the source.To confirm each of the assignations we examined all the aligned reads with BLAST 2.14.1 [94] using the whole NCBI nt dataset, the assigned hits were examined with the LCA algorithm from MEGAN 6.23.3 [95] to confirm the assignations and discard cross-mappings.
Only samples which yielded more than 500 mammalian aDNA reads were further analysed.For these, we generated a consensus sequence with ANGSD [96].The consensus sequences were aligned with other present-day and modern animal sequences with Clustal Omega 1.2.4 [97], as performed in multiple projects assessing the mtDNA diversity of Pleistocene fauna [98][99][100][101] and we then performed a Maximum likelihood (ML) tree with the alignment using MEGA 10.2.4 [102] with partial deletion and 100 bootstrap replications, 95 % partial deletion and GTR substitution model.All trees were plotted with MEGA.

Fig. 2 .
Fig. 2. Scatter plot representing the samples included in the analyses.Y axis represents the target's coverage depth, and the X axis represents the fraction of aligned reads of each sample corresponding to the target.Samples from bone tend to have higher fractions of recovered reads; however, the samples with the highest depth of coverage are all from antlers.

Table 1
Description of the samples.

Table 3
Comparison of minimally-invasive method with the traditional drilling.